Calcium (Ca2+) is an important cellular second messenger and is crucial for many biological phenomena including cell proliferation, bone formation, neuronal excitability, muscle contraction, enzymatic activity, and apoptosis. An important regulator of Ca2+ homeostasis and dietary Ca2+ absorption is TRPV6 (transient receptor potential vanilloid subtype member 6). TRPV6 is constitutively active, is one of only two highly Ca2+ selective ion channels among the TRP superfamily, and mediates the uptake of Ca2+ in epithelial tissues. In addition to its role in Ca2+ uptake in epithelial tissues, the expression of TRPV6 has been implicated in numerous diseases, including breast and prostate cancer, and therefore, has emerged as a target for cancer therapeutics. Additionally, altered TRPV6 expression has been observed in transgenic mouse models of human diseases related to the kidney and bowel, and in the placenta of women suffering from preeclampsia. The lipid compositions of membranes into which TRPV6 ion channels are inserted are responsible for the regulation of TRPV6 channels. However, the structural basis of this regulation is unknown. We will characterize the structure of TRPV6 in artificial lipid bilayers with engineered lipid compositions. To do so, we will use recently developed technologies in cryo-electron microscopy (cryo-EM). The resulting structures will inform not only fundamental biological phenomena, such as dietary Ca2+ absorption and the mechanism by which constitutively active ion channels are regulated, but also will contribute to structure-based drug design targeting TRPV6. Additionally, we will investigate the structural basis of calmodulin (CaM) inactivation of TRPV6. This will provide valuable insight into how CaM might regulate the TRP superfamily of ion channels and tetrameric ion channels in general. All of our structural analyses will be complemented by electrophysiological experiments, especially by recording currents from single ion channels, a technique capable of resolving the kinetics of a single channel as its regulated by agonists and antagonists. !